Skeletal viability requires precise regulation of bone synthesis throughout life, and the catastrophic pathological conditions that accompany its failure present a major public health challenge. Nevertheless, the mechanisms through which synthesis of bone is regulated by signals from the surrounding tissues are not understood, and this lack of knowledge hampers the ability to treat diseases of defective bone growth. The long-term goal of this group is therefore to understand the molecular mechanisms that control differentiation of the osteoblast (OB), the bone-forming cell, so as to better manipulate bone synthesis in the treatment of bone diseases. Recent work in the literature and preliminary data presented herein suggest and support a central role for the contact-mediated promotion of OB differentiation by members of the Eph family of receptor tyrosine kinases and their ligands, the ephrins. Specifically:1) Ephs and ephrins are expressed in discrete layers of osteogenesis in vivo, and 2) treatment of OBs with soluble recombinant ephrin increases OB-specific gene expression and bone synthesis. Based on this evidence, this proposal advances the central hypothesis that Eph signaling regulates bone growth through the activation of specific osteoblast gene promoter elements. The rationale for the proposed studies is that a deeper understanding of how these receptors participate in contact-mediated OB differentiation will reveal control points for exploitation in future therapies. This hypothesis will be addressed through completion of two specific aims:1) Determine the effect of activating or blocking Eph signaling on normal and defective bone formation, and 2) Define the contribution of specific promoter elements to OB gene activation. Studies under the first aim will apply recombinant ephrins to a well-established calvarial model of bone growth to stimulate osteogenesis. Conversely, application of Eph extracellular domain protein will be used to competitively inhibit endogenous ephrins and thus block osteogenesis. These studies have both basic science and translational relevance in that they will establish the importance of, and potential application for, manipulation of Eph/ephrin signaling in the treatment of bone defects. Studies under the second aim will use previously characterized OB-specific promoter-driven luciferase reporters to identify the cis-acting elements within these promoters that are responsible for ephrin induction of gene transcription in OBs. This will lead to identification of transcription factors that may be amenable to manipulation in a clinical setting. The proposed work is innovative because it will uncover an entirely new role for Ephs in gene regulation, and will illuminate a novel mechanism for the regulation of osteogenesis. It will make a significant impact because of its potential to offer new therapeutic targets for the treatment of bone diseases. Public Health Relevance: The planned studies propose a heretofore-undescribed role for Eph/ephrin signaling in the regulation of gene expression. They have particular application to the public health in that they address basic mechanisms of osteoblast differentiation that are dysregulated in the pathogenesis of crippling bone diseases. Thus, they can be expected to contribute to advances in clinical treatment of these afflictions. Further, as ephrins are involved in virtually every stage of organogenesis, this work has the potential for broader impact on the fields of developmental and molecular biology.